Anti-PD-L1 antibody [CAL10]

22 product images

• 

  Western blot - Anti-PD-L1 antibody [CAL10] (ab237726)

  False colour image of Western blot: Anti-PD-L1 antibody [CAL10] - Mouse IgG1 staining at 1/1000 dilution, shown in green; Rabbit anti-alpha Tubulin antibody [EP1332Y] (Anti-alpha Tubulin antibody [EP1332Y] - Loading Control ab52866) loading control staining at 1/20000 dilution, shown in red. In Western blot, Anti-PD-L1 antibody [CAL10] - Mouse IgG1 (Chimeric) ab279292 was shown to bind specifically to PD-L1. A band was observed at 48 kDa in treated wild-type A549 cell lysates with no signal observed at this size in Cd274 knockout cell line Human CD274 (PD-L1) knockout A549 cell line ab267054 (knockout cell lysate Human CD274 (PD-L1) knockout A549 cell lysate ab256831). To generate this image, wild-type and Cd274 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed ab216772) and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed (Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed ab216777) at 1/20000 dilution.

  Lanes 1 - 2: Western blot - Anti-PD-L1 antibody [CAL10] - Mouse IgG1 (Chimeric) (Anti-PD-L1 antibody [CAL10] - Mouse IgG1 (Chimeric) ab279292) at 1/1000 dilution

  Lanes 1 - 2: Western blot - Anti-PD-L1 antibody [CAL10] (ab237726) at 1/1000 dilution

  Lane 1: Wild-type A549 Treated IFN-gamma (100 ng/ml) for 48 hours cell lysate at 20 µg

  Lane 2: CD274 knockout A549 Treated IFN-gamma (100 ng/ml) for 48 hours cell lysate at 20 µg

  Lane 2: Western blot - Human CD274 (PD-L1) knockout A549 cell line (Human CD274 (PD-L1) knockout A549 cell line ab267054)

  Performed under reducing conditions.

  Predicted band size: 33 kDa

  Observed band size: 48 kDa

• 

  Western blot - Anti-PD-L1 antibody [CAL10] (ab237726)

  Blocking/Dilution buffer: 5% NFDM/TBST.

  All lanes: Western blot - Anti-PD-L1 antibody [CAL10] (ab237726) at 1/1000 dilution

  Lane 1: CHO-S (Chinese hamster ovary epithelial cell) whole cell lysate at 20 µg

  Lane 2: CHO-PD-L1 (PD-L1 stably expressed Chinese hamster ovary epithelial cell) whole cell lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Predicted band size: 33 kDa

  Exposure time: 3s

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [CAL10] (ab237726)

  IHC image of ab237726 staining PD-L1 in PD-L1 Dynamic Range Analyte Control formalin fixed paraffin embedded cell lines (HistoCyte Laboratories), performed on a Leica BOND RX (standard Protocol F, Polymer Refine kit). The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 30 mins at 98°C. The section was then incubated with ab237726, 1μg/ml working concentration, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system for 8 minutes at room temperature. DAB was used as the chromogen for 10 minutes at room temperature. The section was then counterstained with hematoxylin, blued, dehydrated, cleared and mounted with DPX.
  

  For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

• 

  Immunocytochemistry/ Immunofluorescence - Anti-PD-L1 antibody [CAL10] (ab237726)

  Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized CHO-PD-L1 (PD-L1 stably expressed Chinese hamster ovary epithelial cell) and CHO-S (chinese hamster ovary epithelial cell) cells labeling PD-L1 with ab237726 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing membranous and cytoplasmic staining in CHO-PD-L1 cells. The nuclear counter stain is DAPI (blue). Counterstained with Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at a 1/200 dilution (red). The negative control is the secondary antibody only.

• 

  Multiplex immunohistochemistry - Anti-PD-L1 antibody [CAL10] (ab237726)

  PD-L1 Multiplex immunohistochemistry staining of human tonsil tissue using rabbit Anti-PD-L1 antibody

  Fluorescence multiplex immunohistochemical analysis of normal human tonsil tissue (formalin-fixed paraffin-embedded section).

  Merged staining of anti-PD1 (Anti-PD1 antibody [CAL20] ab237728; orange; Opal™520) anti-PDL1 (ab237726; green; Opal™540) anti-CD68 (Anti-CD68 antibody [SP251] ab192847; yellow; Opal™570) anti-CD3 epsilon (Anti-CD3 epsilon antibody [SP7] ab16669; red; Opal™620) anti-Ki67 (Anti-Ki67 antibody [SP6] ab16667; light blue; Opal™650) and anti-PanCK (Anti-pan Cytokeratin antibody [C-11] ab7753; grey; Opal™690).

  The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with an Opal™ 7-color automation IHC kit (NEL821001KT Akoya Biosciences^®).

  The section was incubated in six rounds of staining; in the order of Anti-PD1 antibody [CAL20] ab237728 (1/500 dilution), ab237726 (1/500 dilution), Anti-CD68 antibody [SP251] ab192847 (1/300 dilution), Anti-CD3 epsilon antibody [SP7] ab16669 (1/300 dilution), Anti-Ki67 antibody [SP6] ab16667 (1/200 dilution) and Anti-pan Cytokeratin antibody [C-11] ab7753 (1/200 dilution); each using a separate fluorescent tyramide signal amplification system.

  Sodium citrate antigen retrieval (Leica ER1 pH6.0 30 minutes) was used in between rounds of tyramide signal amplification to remove the antibody from the previous round to avoid any cross-reactivity.

  DAPI (dark blue) was used as a nuclear counter stain.

  Microscopy and pseudocoloring of individual Opal™ dyes was performed using a Vectra Polaris.

• 

  Western blot - Anti-PD-L1 antibody [CAL10] (ab237726)

  Blocking/Dilution buffer: 5% NFDM/TBST.
  

  The molecular mass observed is consistent with what has been described in the literature (PMID: 26546452).

  All lanes: Western blot - Anti-PD-L1 antibody [CAL10] (ab237726) at 1/1000 dilution

  Lane 1: A549 (Human lung carcinoma epithelial cell) whole cell lysate at 20 µg

  Lane 2: A549 treated with 100ng/ml IFN gamma for 48h whole cell lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Predicted band size: 33 kDa

  Exposure time: 26s

• 

  Immunoprecipitation - Anti-PD-L1 antibody [CAL10] (ab237726)

  PD-L1 was immunoprecipitated from 0.35 mg NCI-H1975 (human non-small cell lung cancer cell line) whole cell lysate with ab237726 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab237726 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366), was used at 1/5000 dilution.
  Lane 1: NCI-H1975 whole cell lysate 10 μg (Input).
  Lane 2: ab237726 IP in NCI-H1975 whole cell lysate.
  Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab237726 in NCI-H1975 whole cell lysate.
  Blocking/Dilution buffer: 5% NFDM/TBST.
  Exposure time: 30 seconds.
  

  The molecular mass observed is consistent with what has been described in the literature (PMID: 26546452).

  All lanes: Immunoprecipitation - Anti-PD-L1 antibody [CAL10] (ab237726)

  Predicted band size: 33 kDa

• 

  Western blot - Anti-PD-L1 antibody [CAL10] (ab237726)

  Blocking/Dilution buffer: 5% NFDM/TBST.

  All lanes: Western blot - Anti-PD-L1 antibody [CAL10] (ab237726) at 1/1000 dilution

  All lanes: Human placenta lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Predicted band size: 33 kDa

  Exposure time: 26s

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [CAL10] (ab237726)

  Immunohistochemical analysis of paraffin-embedded human lung carcinoma tissue labeling PD-L1 with ab237726 at 1/1000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Mainly membranous staining on the human lung carcinoma. The section was incubated with ab237726 for 15 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin. Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 30 mins.

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [CAL10] (ab237726)

  Immunohistochemical analysis of paraffin-embedded human placenta tissue labeling PD-L1 with ab237726 at 1/1000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Membranous staining on the human placenta. The section was incubated with ab237726 for 15 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin. Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 30 mins.

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [CAL10] (ab237726)

  Formalin-fixed, paraffin-embedded NSCLC (Non-small-cell lung carcinoma) tissue stained for PD L1 using ab237726 at 0.3 μg/ml dilution in immunohistochemical analysis. Positive staining (Left panel) and negative staining (Right panel).

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [CAL10] (ab237726)

  Formalin-fixed, paraffin-embedded PD L1 transfected HEK-293 (Human epithelial cell line from embryonic kidney) cells stained for PD L1 using ab237726 at 0.3 μg/ml dilution (Left panel) in immunohistochemical analysis.
  

  Negative control (Right panel): PD L2 transfected HEK-293 cells.

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  ELISA - Anti-PD-L1 antibody [CAL10] (ab237726)

  ELISA - Anti-PD-L1 antibody [CAL10] (ab237726).
• 

  This image is courtesy of ImmunoAtlas.

  Multiplex immunohistochemistry - Anti-PD-L1 antibody [CAL10] (ab237726)

  Fluorescence multiplex immunohistochemical analysis of Human breast cancer tissue (formalin-fixed paraffin-embedded section).
  

  Merged staining of Anti-PD-L1 (Anti-PD-L1 antibody [CAL10] - BSA and Azide free ab251611; cyan; Opal™ 520), Anti-Granzyme B (Anti-Granzyme B antibody [EPR20129-217] - BSA and Azide free ab219803; yellow; Opal™ 540), Anti-PD1 (Anti-PD1 antibody [CAL20] - BSA and Azide free ab251613; magenta; Opal™ 570), Anti-pan Cytokeratin (Anti-pan Cytokeratin antibody [C-11] - BSA and Azide free ab264485; red; Opal™ 620), Anti-EpCAM (Anti-EpCAM antibody [EPR20532-225] - BSA and Azide free ab225894; red; Opal™ 620), Anti-CD8 alpha (Anti-CD8 alpha antibody [CAL66] - BSA and Azide free ab251596; green; Opal™ 650) and Anti-FOXP3 (Anti-FOXP3 antibody [236A/E7] - BSA and Azide free ab96048; orange; Opal™ 690). EpCAM and pan-cytokeratin share the same dye and color. Dyes are pseudo-colored for better contrast of the markers.

  The immunostaining was performed on a Leica Biosystems BOND^® MAX instrument with an Opal™ 6-Plex Detection Kit (NEL821001KT, Akoya Biosciences^®).

  The section was incubated in six rounds of staining; sequentially for Anti-PD-L1 antibody [CAL10] - BSA and Azide free ab251611 (1/750 dilution), Anti-Granzyme B antibody [EPR20129-217] - BSA and Azide free ab219803 (1/250 dilution), Anti-PD1 antibody [CAL20] - BSA and Azide free ab251613 (1/750 dilution), Anti-pan Cytokeratin antibody [C-11] - BSA and Azide free ab264485 (0.5 μg/ml), Anti-EpCAM antibody [EPR20532-225] - BSA and Azide free ab225894 (1/1250 dilution), Anti-CD8 alpha antibody [CAL66] - BSA and Azide free ab251596 (1/1500 dilution) and Anti-FOXP3 antibody [236A/E7] - BSA and Azide free ab96048 (10 μg/ml); each using a separate fluorescent tyramide signal amplification system. EDTA based antigen retrieval (Leica Biosystems BOND^® Epitope Retrieval Solution 2, pH 9.0, 20 minutes) was used in between rounds of tyramide signal amplification to remove the antibody from the previous round, to avoid any cross-reactivity. DAPI (dark blue) was used as a nuclear counter stain.
  Microscopy and pseudocoloring of individual Opal™ dyes was performed using a Vectra 3 Imaging System (Akoya Biosciences^®).

  This data is courtesy of ImmunoAtlas and it can be found here.

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [CAL10] (ab237726)

  Tissue Microarrays stained for "Anti-PD-L1 antibody [CAL10]" using "ab237726"in immunohistochemical analysis. This table provides a detailed overview of positive (tick mark) and negative (cross mark) staining per sample type tested. The sections were pre-treated using Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0) for 30 minutes. The sections were incubated with ab237726 for 15 mins at room temperature followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

• 

  Western blot - Anti-PD-L1 antibody [CAL10] (ab237726)

  Anti-PD-L1 antibody [73-10] ab228415 works better than ab237726 in western blot testing.
  We suggest optimizing experimental protocols (increasing lysate amount, using lower dilution or higher sensitivity ECL substrate) to improve results.
  
  Blocking/Diluting buffer and concentration: 5% NFDM/TBST

  Lanes 1 - 4: Western blot - Anti-PD-L1 antibody [CAL10] (ab237726) at 1/1000 dilution

  Lanes 1 - 4: Western blot - Anti-PD-L1 antibody [CAL10] - BSA and Azide free (Anti-PD-L1 antibody [CAL10] - BSA and Azide free ab251611)

  Lane 1: PC-3 (human prostate adenocarcinoma epithelial cell) whole cell lysate at 20 µg

  Lane 2: A375 (human malignant melanoma epithelial cell) whole cell lysate at 20 µg

  Lane 3: HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

  Lane 4: A549 (human lung carcinoma epithelial cell) whole cell lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Observed band size: 40-60 kDa

  Exposure time: 100s

• 

  This image is courtesy of ImmunoAtlas.

  Multiplex immunohistochemistry - Anti-PD-L1 antibody [CAL10] (ab237726)

  Fluorescence multiplex immunohistochemical analysis of Human breast cancer tissue (formalin-fixed paraffin-embedded section).
  

  Merged staining of Anti-PD-L1 (Anti-PD-L1 antibody [CAL10] - BSA and Azide free ab251611; cyan; Opal™ 520), Anti-Granzyme B (Anti-Granzyme B antibody [EPR20129-217] - BSA and Azide free ab219803; yellow; Opal™ 540), Anti-PD1 (Anti-PD1 antibody [CAL20] - BSA and Azide free ab251613; magenta; Opal™ 570), Anti-pan Cytokeratin (Anti-pan Cytokeratin antibody [C-11] - BSA and Azide free ab264485; red; Opal™ 620), Anti-EpCAM (Anti-EpCAM antibody [EPR20532-225] - BSA and Azide free ab225894; red; Opal™ 620), Anti-CD8 alpha (Anti-CD8 alpha antibody [CAL66] - BSA and Azide free ab251596; green; Opal™ 650) and Anti-FOXP3 (Anti-FOXP3 antibody [236A/E7] - BSA and Azide free ab96048; orange; Opal™ 690). EpCAM and pan-cytokeratin share the same dye and color. Dyes are pseudo-colored for better contrast of the markers.

  The immunostaining was performed on a Leica Biosystems BOND^® MAX instrument with an Opal™ 6-Plex Detection Kit (NEL821001KT, Akoya Biosciences^®).

  The section was incubated in six rounds of staining; sequentially for Anti-PD-L1 antibody [CAL10] - BSA and Azide free ab251611 (1/750 dilution), Anti-Granzyme B antibody [EPR20129-217] - BSA and Azide free ab219803 (1/250 dilution), Anti-PD1 antibody [CAL20] - BSA and Azide free ab251613 (1/750 dilution), Anti-pan Cytokeratin antibody [C-11] - BSA and Azide free ab264485 (0.5 μg/ml), Anti-EpCAM antibody [EPR20532-225] - BSA and Azide free ab225894 (1/1250 dilution), Anti-CD8 alpha antibody [CAL66] - BSA and Azide free ab251596 (1/1500 dilution) and Anti-FOXP3 antibody [236A/E7] - BSA and Azide free ab96048 (10 μg/ml); each using a separate fluorescent tyramide signal amplification system. EDTA based antigen retrieval (Leica Biosystems BOND^® Epitope Retrieval Solution 2, pH 9.0, 20 minutes) was used in between rounds of tyramide signal amplification to remove the antibody from the previous round, to avoid any cross-reactivity. DAPI (dark blue) was used as a nuclear counter stain.
  Microscopy and pseudocoloring of individual Opal™ dyes was performed using a Vectra 3 Imaging System (Akoya Biosciences^®).

  This data is courtesy of ImmunoAtlas and it can be found here.

• 

  This image is courtesy of ImmunoAtlas.

  Multiplex immunohistochemistry - Anti-PD-L1 antibody [CAL10] (ab237726)

  Fluorescence multiplex immunohistochemical analysis of Human breast cancer tissue (formalin-fixed paraffin-embedded section).
  

  Merged staining of Anti-PD-L1 (Anti-PD-L1 antibody [CAL10] - BSA and Azide free ab251611; cyan; Opal™ 520), Anti-Granzyme B (Anti-Granzyme B antibody [EPR20129-217] - BSA and Azide free ab219803; yellow; Opal™ 540), Anti-PD1 (Anti-PD1 antibody [CAL20] - BSA and Azide free ab251613; magenta; Opal™ 570), Anti-pan Cytokeratin (Anti-pan Cytokeratin antibody [C-11] - BSA and Azide free ab264485; red; Opal™ 620), Anti-EpCAM (Anti-EpCAM antibody [EPR20532-225] - BSA and Azide free ab225894; red; Opal™ 620), Anti-CD8 alpha (Anti-CD8 alpha antibody [CAL66] - BSA and Azide free ab251596; green; Opal™ 650) and Anti-FOXP3 (Anti-FOXP3 antibody [236A/E7] - BSA and Azide free ab96048; orange; Opal™ 690). EpCAM and pan-cytokeratin share the same dye and color. Dyes are pseudo-colored for better contrast of the markers.

  The immunostaining was performed on a Leica Biosystems BOND^® MAX instrument with an Opal™ 6-Plex Detection Kit (NEL821001KT, Akoya Biosciences^®).

  The section was incubated in six rounds of staining; sequentially for Anti-PD-L1 antibody [CAL10] - BSA and Azide free ab251611 (1/750 dilution), Anti-Granzyme B antibody [EPR20129-217] - BSA and Azide free ab219803 (1/250 dilution), Anti-PD1 antibody [CAL20] - BSA and Azide free ab251613 (1/750 dilution), Anti-pan Cytokeratin antibody [C-11] - BSA and Azide free ab264485 (0.5 μg/ml), Anti-EpCAM antibody [EPR20532-225] - BSA and Azide free ab225894 (1/1250 dilution), Anti-CD8 alpha antibody [CAL66] - BSA and Azide free ab251596 (1/1500 dilution) and Anti-FOXP3 antibody [236A/E7] - BSA and Azide free ab96048 (10 μg/ml); each using a separate fluorescent tyramide signal amplification system. EDTA based antigen retrieval (Leica Biosystems BOND^® Epitope Retrieval Solution 2, pH 9.0, 20 minutes) was used in between rounds of tyramide signal amplification to remove the antibody from the previous round, to avoid any cross-reactivity. DAPI (dark blue) was used as a nuclear counter stain.
  Microscopy and pseudocoloring of individual Opal™ dyes was performed using a Vectra 3 Imaging System (Akoya Biosciences^®).

  This data is courtesy of ImmunoAtlas and it can be found here.

• 

  This image is courtesy of ImmunoAtlas.

  Multiplex immunohistochemistry - Anti-PD-L1 antibody [CAL10] (ab237726)

  Fluorescence multiplex immunohistochemical analysis of Human breast cancer tissue (formalin-fixed paraffin-embedded section).
  

  Merged staining of Anti-PD-L1 (Anti-PD-L1 antibody [CAL10] - BSA and Azide free ab251611; cyan; Opal™ 520), Anti-Granzyme B (Anti-Granzyme B antibody [EPR20129-217] - BSA and Azide free ab219803; yellow; Opal™ 540), Anti-PD1 (Anti-PD1 antibody [CAL20] - BSA and Azide free ab251613; magenta; Opal™ 570), Anti-pan Cytokeratin (Anti-pan Cytokeratin antibody [C-11] - BSA and Azide free ab264485; red; Opal™ 620), Anti-EpCAM (Anti-EpCAM antibody [EPR20532-225] - BSA and Azide free ab225894; red; Opal™ 620), Anti-CD8 alpha (Anti-CD8 alpha antibody [CAL66] - BSA and Azide free ab251596; green; Opal™ 650) and Anti-FOXP3 (Anti-FOXP3 antibody [236A/E7] - BSA and Azide free ab96048; orange; Opal™ 690). EpCAM and pan-cytokeratin share the same dye and color. Dyes are pseudo-colored for better contrast of the markers.

  The immunostaining was performed on a Leica Biosystems BOND® MAX instrument with an Opal™ 6-Plex Detection Kit (NEL821001KT, Akoya Biosciences^®).

  The section was incubated in six rounds of staining; sequentially for Anti-PD-L1 antibody [CAL10] - BSA and Azide free ab251611 (1/750 dilution), Anti-Granzyme B antibody [EPR20129-217] - BSA and Azide free ab219803 (1/250 dilution), Anti-PD1 antibody [CAL20] - BSA and Azide free ab251613 (1/750 dilution), Anti-pan Cytokeratin antibody [C-11] - BSA and Azide free ab264485 (0.5 μg/ml), Anti-EpCAM antibody [EPR20532-225] - BSA and Azide free ab225894 (1/1250 dilution), Anti-CD8 alpha antibody [CAL66] - BSA and Azide free ab251596 (1/1500 dilution) and Anti-FOXP3 antibody [236A/E7] - BSA and Azide free ab96048 (10 μg/ml); each using a separate fluorescent tyramide signal amplification system. EDTA based antigen retrieval (Leica Biosystems BOND^® Epitope Retrieval Solution 2, pH 9.0, 20 minutes) was used in between rounds of tyramide signal amplification to remove the antibody from the previous round, to avoid any cross-reactivity. DAPI (dark blue) was used as a nuclear counter stain.
  Microscopy and pseudocoloring of individual Opal™ dyes was performed using a Vectra 3 Imaging System (Akoya Biosciences^®).

  This data is courtesy of ImmunoAtlas and it can be found here.

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [CAL10] (ab237726)

  Immunohistochemical analysis of formalin-fixed paraffin-embedded human placenta labelling PD-L1 with ab237726 at a dilution of 2µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with an OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was conducted for 32min with ULTRA cell conditioning solution (CC1 pH8.5). ab237726 anti PD-L1 antibody was incubated at 37°C for 16min. Sections were counterstained is with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PD-L1 antibody [CAL10] (ab237726)

  Immunohistochemical analysis of formalin-fixed paraffin-embedded human tonsil labelling PD-L1 with ab237726 at a dilution of 1µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with an OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was conducted for 32min with ULTRA cell conditioning solution (CC1 pH8.5). ab237726 anti PD-L1 antibody was incubated at 37°C for 16min. Sections were counterstained is with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

• 

  This image is courtesy of ImmunoAtlas.

  Multiplex immunohistochemistry - Anti-PD-L1 antibody [CAL10] (ab237726)

  Fluorescence multiplex immunohistochemical analysis of Human breast cancer tissue (formalin-fixed paraffin-embedded section).
  

  Merged staining of Anti-PD-L1 (Anti-PD-L1 antibody [CAL10] - BSA and Azide free ab251611; cyan; Opal™ 520), Anti-Granzyme B (Anti-Granzyme B antibody [EPR20129-217] - BSA and Azide free ab219803; yellow; Opal™ 540), Anti-PD1 (Anti-PD1 antibody [CAL20] - BSA and Azide free ab251613; magenta; Opal™ 570), Anti-pan Cytokeratin (Anti-pan Cytokeratin antibody [C-11] - BSA and Azide free ab264485; red; Opal™ 620), Anti-EpCAM (Anti-EpCAM antibody [EPR20532-225] - BSA and Azide free ab225894; red; Opal™ 620), Anti-CD8 alpha (Anti-CD8 alpha antibody [CAL66] - BSA and Azide free ab251596; green; Opal™ 650) and Anti-FOXP3 (Anti-FOXP3 antibody [236A/E7] - BSA and Azide free ab96048; orange; Opal™ 690). EpCAM and pan-cytokeratin share the same dye and color. Dyes are pseudo-colored for better contrast of the markers.

  The immunostaining was performed on a Leica Biosystems BOND^® MAX instrument with an Opal™ 6-Plex Detection Kit (NEL821001KT, Akoya Biosciences^®).

  The section was incubated in six rounds of staining; sequentially for Anti-PD-L1 antibody [CAL10] - BSA and Azide free ab251611 (1/750 dilution), Anti-Granzyme B antibody [EPR20129-217] - BSA and Azide free ab219803 (1/250 dilution), Anti-PD1 antibody [CAL20] - BSA and Azide free ab251613 (1/750 dilution), Anti-pan Cytokeratin antibody [C-11] - BSA and Azide free ab264485 (0.5 μg/ml), Anti-EpCAM antibody [EPR20532-225] - BSA and Azide free ab225894 (1/1250 dilution), Anti-CD8 alpha antibody [CAL66] - BSA and Azide free ab251596 (1/1500 dilution) and Anti-FOXP3 antibody [236A/E7] - BSA and Azide free ab96048 (10 μg/ml); each using a separate fluorescent tyramide signal amplification system. EDTA based antigen retrieval (Leica Biosystems BOND^® Epitope Retrieval Solution 2, pH 9.0, 20 minutes) was used in between rounds of tyramide signal amplification to remove the antibody from the previous round, to avoid any cross-reactivity. DAPI (dark blue) was used as a nuclear counter stain.
  Microscopy and pseudocoloring of individual Opal™ dyes was performed using a Vectra 3 Imaging System (Akoya Biosciences^®).

  This data is courtesy of ImmunoAtlas and it can be found here.